Volume 10, Issue 3, Pages 703-711 (March 2018) Calorie Restriction Governs Intestinal Epithelial Regeneration through Cell-Autonomous Regulation of mTORC1 in Reserve Stem Cells  Maryam Yousefi, Angela Nakauka-Ddamba, Corbett T. Berry, Ning Li, Jenna Schoenberger, Kamen P. Simeonov, Ryan J. Cedeno, Zhengquan Yu, Christopher J. Lengner  Stem Cell Reports  Volume 10, Issue 3, Pages 703-711 (March 2018) DOI: 10.1016/j.stemcr.2018.01.026 Copyright © 2018 The Author(s) Terms and Conditions

Stem Cell Reports 2018 10, 703-711DOI: (10.1016/j.stemcr.2018.01.026) Copyright © 2018 The Author(s) Terms and Conditions

Figure 1 Calorie Restriction Expands the Pool of Radioresistant Reserve Intestinal Stem Cells (A) Immunofluorescence staining for tdTomato (red) on jejunal sections from Hopx-CreER::LSL-tdTomato ad libitum (AL)-fed and calorie-restricted (CR) mice 18 hr after a single tamoxifen injection. Epithelial crypts are outlined by a white dashed line (n = 4–6 mice). Scale bar, 50 μm. (B) Analysis of reserve ISC frequency in CR and AL-fed Hopx-CreER::LSL-tdTomato and Bmi1-CreER::LSL-tdTomato mice using flow cytometry, 18 hr after tamoxifen injection (n = 4–6 mice, ∗p < 0.05). Details of gating strategy can be found in Figure S1A. (C) Analysis of EdU incorporation in reserve ISCs and crypt cells of AL-fed and CR Hopx-CreER::LSL-tdTomato and Bmi1-CreER::LSL-tdTomato mice using flow cytometry, 18 hr after tamoxifen injection and 4 hr after EdU injection. The proliferation of CR groups is normalized to their control AL-fed counterparts (n = 4–6 mice, ∗p < 0.05 and ∗∗∗p < 0.0005). (D) Immunofluorescence staining for tdTomato (red) and quantification of lineage-tracing events (ribbons of tdTomato+ cells with contiguous tracing from crypts, through crypt-villus junction, and into villi) from Hopx-CreER+ ISCs 14 days after marking reserve ISCs with a single tamoxifen injection given to Hopx-CreER::LSL-tdTomato mice under AL and CR diets (n = 5–6 mice, ∗p < 0.05), Scale bar, 100 μm. (E) Representative Ki67(red)-stained sections from jejunum of irradiated Hopx-CreER::LSL-DTA mice and their control counterparts (LSL-DTA) mice after 6 weeks of receiving CR or AL diet and quantification of the number of clonal proliferative crypt foci per unit length of small intestine. All groups of mice were given three daily consecutive doses of tamoxifen and were exposed to 12 Gy γ-IR 3 days after the final dose. Tissue was harvested 3 days after γ-IR (n = 5 mice, ∗∗∗p < 0.0005). Scale bar, 100 μm. Error bars represent ±SD from the mean. See also Figures S1 and S2. Stem Cell Reports 2018 10, 703-711DOI: (10.1016/j.stemcr.2018.01.026) Copyright © 2018 The Author(s) Terms and Conditions

Figure 2 mTORC1 Activity Is Precisely Regulated during the Regenerative Response of the Intestinal Epithelium following Radiation Injury (A) GSEA of the reserve ISC transcriptome isolated from Hopx-CreER::LSL-tdTomato mice under AL versus CR conditions. (B and C) Immunofluorescence staining for pS6 (red) in the jejunum of CR and AL-fed mice under basal conditions (pre-IR) and 3 days after radiation injury. The exposure time in (B) is higher (5 s) than that in the top panels of (C) (1 s) in order to clearly highlight differences in pS6 levels under basal conditions (n = 5 mice). Scale bar, 50 μm. (D) Immunofluorescence staining for pS6 and quantification of pS6 fluorescence intensity in single reserve ISCs from Hopx-CreER::LSL-tdTomato AL-fed and CR mice, 18 hr after tamoxifen injection (n = 3 mice, 30–40 ISCs scored per animal, ∗∗p < 0.005). Scale bar, 5 μm. (E) Western blot analysis for pS6 in FACS-purified reserve stem cells from Hopx-CreER::LSL-tdTomato mice under basal conditions and 48 hr after irradiation injury. Both groups were given tamoxifen 18 hr before tissue harvest. In total, 21,000 cells were loaded per lane (representative blot from n = 3 mice). See also Figure S3 and Table S1. Stem Cell Reports 2018 10, 703-711DOI: (10.1016/j.stemcr.2018.01.026) Copyright © 2018 The Author(s) Terms and Conditions

Figure 3 mTORC1 Modulates Activation of Reserve ISCs in a Cell-Autonomous Manner (A) Western blot analysis for pS6 in protein extracted from the intestinal epithelium of a cohort of mice that were fasted for 18 hr and then divided into two groups. One group was harvested after fasting and the other group received 1.5% leucine in the drinking water for 6 hr following fasting (n = 3 mice). (B) Lineage tracing from Hopx-CreER+ ISCs using immunofluorescence staining for tdTomato in sections from Hopx-CreER::LSL-tdTomato and control mice 2 weeks after tamoxifen treatment. Rapamycin and leucine treatments started 3 days before tamoxifen injection and continued until time of harvest (n = 3–5 mice, ∗∗∗p < 0.0005). Scale bar, 100 μm. (C) Organoid formation assays from single FACS-purified Hopx-CreER+ ISCs from Hopx-CreER::LSL-tdTomato mice that received one dose of tamoxifen 18 hr before tissue harvest. Sorted cells were treated with 10 μM leucine or rapamycin for 72 hr in culture (n = 3 replicate wells from each of two individual animals, ∗p < 0.05 and ∗∗p < 0.005). Images of organoid cultures are presented in Figure S3B. (D) Lineage tracing from reserve ISCs in Hopx-CreER::Tsc1flox/flox::LSL-tdTomato (THT) mice visualized with immunofluorescence staining for tdTomato in sections from the jejunum 2 weeks after tamoxifen treatment (n = 5 mice, ∗p < 0.05). Scale bar, 100 μm. Error bars represent ±SD from the mean. See also Figure S3. Stem Cell Reports 2018 10, 703-711DOI: (10.1016/j.stemcr.2018.01.026) Copyright © 2018 The Author(s) Terms and Conditions

Figure 4 Premature Activation of mTORC1 Sensitizes Reserve ISCs to Radiation Injury and Results in Regeneration Failure (A) Flow-cytometric analysis of apoptosis in reserve ISCs from Hopx-CreER::LSL-tdTomato mice by staining for cleaved caspase-3, 2 hr after 12 Gy γ-IR. Rapamycin and leucine treatments were started 3 days before the time of harvest, and all groups were given a single tamoxifen dose 18 hr prior to tissue harvest (n = 7–10 mice, ∗∗∗p < 0.0005). (B) Ki67(red) staining and quantification of the regeneration efficiency after irradiation injury in mice receiving 1.5% leucine in their drinking water 3 days before γ-IR and control groups. Arrowheads point to regenerative crypt foci (n = 3 mice, ∗∗p < 0.005). Scale bar, 50 μm. (C) Integrated model describing changes in the radioresistant reserve ISC pool in response to dietary modulation. CR increases the pool of reserve ISCs, while leucine stimulation drives activation of dormant ISCs (dormant: mTORC1-low, orange; active: mTORC1-high, green). Upon exposure to DNA-damaging injury, mTORC1-high ISCs undergo apoptosis. After injury, the surviving pool of ISCs is larger under CR, enabling a more robust regenerative response relative to AL-fed counterparts. In contrast, after leucine feeding, fewer ISCs survive the injury, resulting in regenerative failure. Error bars represent ±SD from the mean. See also Figure S4. Stem Cell Reports 2018 10, 703-711DOI: (10.1016/j.stemcr.2018.01.026) Copyright © 2018 The Author(s) Terms and Conditions